LOCUS       sequence-147436-        5374 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Sacsin HEPN domain (4441-4579) in fusion with GST 
            (bacterial).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5374)
  AUTHORS   Kozlov G, Denisov AY, Girard M, Dicaire MJ, Hamlin J, McPherson PS, 
            Brais B, Gehring K
  TITLE     Structural basis of defects in the sacsin HEPN domain responsible 
            for autosomal recessive spastic ataxia of Charlevoix-Saguenay 
            (ARSACS).
  JOURNAL   J Biol Chem. 2011 Jun 10;286(23):20407-12. doi: 
            10.1074/jbc.M111.232884. Epub 2011 Apr 20.
  PUBMED    21507954
REFERENCE   2  (bases 1 to 5374)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1074/jbc.M111.232884"; journalName: "J Biol Chem"; date: 
            "2011-06-10- 10"; volume: "286"; issue: "23"; pages: "20407-12"
COMMENT     Sequence Label: pGEX4T-HEPN
FEATURES             Location/Qualifiers
     source          1..5374
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(183..188,189..204,205..211)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    219..235
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     CDS             258..911
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     869..891
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             918..935
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(1424..1446)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1591..1609)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1677..1781
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1782..1850,1851..2642)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2000..2019)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2813..3401
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3302..3321
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3555..3572
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3645..3722
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             3723..4805
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(3742..3761)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    4818..4839
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4854..4859,4860..4877,4878..4884)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4892..4908
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     4916..4932
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4916..4932
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(4928..5374,1..81)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGITLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(4948..4965)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4948..4964)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4957..4979)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 acgttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg
       61 gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt
      121 tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc
      181 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca
      241 cacaggaaac agtattcatg tcccctatac taggttattg gaaaattaag ggccttgtgc
      301 aacccactcg acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc
      361 gcgatgaagg tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc
      421 ttccttatta tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata
      481 tagctgacaa gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc
      541 ttgaaggagc ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact
      601 ttgaaactct caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag
      661 atcgtttatg tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt
      721 tgtatgacgc tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa
      781 aattagtttg ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat
      841 ccagcaagta tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc
      901 atcctccaaa atcggatctg gttccgcgtg gatccatggt tggcaatcca gtggaagcac
      961 gcagatggct aagacaagcc agagcaaact tctcagctgc caggaatgac cttcataaaa
     1021 atgccaatga gtgggtgtgc tttaaatgtt acctttctac caagttagct ttgattgcag
     1081 ctgactatgc tgtgagggga aagtctgata aagatgtaaa accaactgca cttgctcaga
     1141 aaatagagga atatagtcag caacttgaag gactgacaaa tgatgttcac acattggaag
     1201 cttatggtgt agacagttta aaaacaagat accctgattt gcttcccttt cctcagatcc
     1261 caaatgacag gttcacttct gaggttgcta tgagggtgat ggaatgtact gcctgtatca
     1321 taataaaact tgaaaatttt atgcaacaaa aagtgtaact cgagcggccg catcgtgact
     1381 gactgacgat ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc
     1441 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg
     1501 gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata
     1561 gcggagtgta taattcttga agacgaaagg gcctcgtgat acgcctattt ttataggtta
     1621 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
     1681 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     1741 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     1801 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     1861 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     1921 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     1981 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtgttgac gccgggcaag
     2041 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     2101 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     2161 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     2221 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     2281 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa
     2341 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     2401 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     2461 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     2521 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     2581 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     2641 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     2701 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     2761 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     2821 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     2881 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     2941 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     3001 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     3061 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     3121 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     3181 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     3241 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     3301 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     3361 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     3421 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     3481 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     3541 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt
     3601 ttctccttac gcatctgtgc ggtatttcac accgcataaa ttccgacacc atcgaatggt
     3661 gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga
     3721 atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg
     3781 tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag
     3841 cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac
     3901 agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg
     3961 tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag
     4021 aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca
     4081 gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct
     4141 gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta
     4201 ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc
     4261 agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg
     4321 gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact
     4381 ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca
     4441 ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt
     4501 ccgggctgcg cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct
     4561 catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca
     4621 gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc
     4681 ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc
     4741 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     4801 agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac
     4861 tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga
     4921 aacagctatg accatgatta cggattcact ggccgtcgtt ttacaacgtc gtgactggga
     4981 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
     5041 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     5101 atggcgcttt gcctggtttc cggcaccaga agcggtgccg gaaagctggc tggagtgcga
     5161 tcttcctgag gccgatactg tcgtcgtccc ctcaaactgg cagatgcacg gttacgatgc
     5221 gcccatctac accaacgtaa cctatcccat tacggtcaat ccgccgtttg ttcccacgga
     5281 gaatccgacg ggttgttact cgctcacatt taatgttgat gaaagctggc tacaggaagg
     5341 ccagacgcga attatttttg atggcgttgg aatt
//
